Project Summary Glycosaminoglycans (GAGs) play important roles in many physiological and pathological events such as cell division, inflammation, neural development, and cancer metastasis. The long polysaccharide chains of GAGs contain various sulfated disaccharides that are organized into sulfate-rich and under-sulfated domains. This rich structural diversity enables GAGs such as heparan sulfate (HS) to interact with numerous proteins and regulate key signaling pathways. However, efforts to understand their structure-function relationships and harness their therapeutic potential have been hampered by the chemical complexity of GAGs and a lack of tools. At present, there are no tools to manipulate the interactions of GAGs with specific proteins of interest, thus complicating efforts to pinpoint their precise roles. The goal of this project is to develop novel chemical probes for selectively modulating GAG activity. We will use a directed evolution-based approach to create a new class of GAG mimetics ? multivalent glycopeptides appended with short, active HS motifs ? to modulate specific HS-protein interactions. Random sampling of peptide sequences by directed evolution should allow for the selection of structures containing the ideal number and arrangement of HS motifs. In addition to optimal HS clustering, selected peptides should contain peptide motifs recognized by the protein of interest, thus generating highly specific GAG probes. In Aim 1, we will work in collaboration with the Krauss laboratory to develop the approach and generate HS mimetics that interact selectively with fibroblast growth factor 2 (FGF2), a key growth factor involved in cell migration, angiogenesis, and differentiation. In Aim 2, we will evolve glycopeptides that bind to specific forms of tau, a microtubule-associated protein linked to dementias such as Alzheimer's disease and Parkinson's disease. We will use these HS mimetics to understand the mechanisms underlying tau uptake into neurons and neurodegeneration. In addition, we will explore whether our mimetics can block the intercellular spreading of tau and its pathogenic consequences. In Aim 3, we will evolve glycopeptides that bind chemokines (specifically CXCL9, CXCL10 and CXCL11), a class of therapeutically important proteins that are key mediators of inflammation. Our probes should provide unique insights into the paradoxical functional redundancy of chemokines, enabling us to tease apart their individual roles. Together, these studies will produce a novel class of GAG-based probes for understanding the physiological functions of GAGs and may ultimately lead to new therapeutic leads or approaches to diseases such as cancer, neurodegenerative diseases, inflammatory and autoimmune disorders, and infectious diseases.